Disturbances in the anionic sites of the retinal microvasculature (RMV) may contribute to the increased capillary permeability characteristic of diabetic retinopathy. Several approaches will be used to examine these anionic sites in RMV basement membranes purified from the retinas of nondiabetic and diabetic subjects. First, the interaction of Platelet Factor 4 (PF4) with glycosaminoglycans (GAG) of RMV matrix will be assessed. PF4 has unique regions of positive charge and a natural affinity for (carrier) proteoglycan; it can bind to heparin, and to heparan sulfate isolated from human renal cortex. These considerations suggest that PF4 can bind to GAG of RMV walls and that, in view of increased PF4 release and endothelial damage in diabetes, this binding influences the permeability of the RMV in diabetes. Second, heparan sulfate and other GAG anionic sites will be directly measured of means of a sensitive competitive-displacement assay. Third, the number and accessibility of anionic sites in isolated retinal microvessel basement membranes will be related to that in intact vessels by examining the incorporation of radiolabeled saccharide into proteoglycan-protein core of the microvessel matrix. Finally, the integrity of the proteoglycan-GAG linkage region in normal vs diabetic preparations will be explored. PF4 will be purified from platelet concentrates and used to determine its binding to sulfated GAG isolated from RMV-BM. The GAG will be characterized by cellulose acetate electrophoresis and susceptibility to specific enzymatic digestion. Complex formation between PF4 and GAG will be assessed with electrophoresis, gel filtration, and 125I-PF4 binding. The latter technique will be adapted to permit measurement of binding, and hence, indirectly, quantification of anionic sites, in RMV-BM purified from non-diabetic and diabetic subjects. Direct analysis of GAG anionic sites in normal and diabetic RMV-BM will be performed by measuring the displacement by tissue digests of 3H-heparan sulfate complexed with serum lipoproteins. Radiolabeled saccharide incorporation will utilize enzymatic methods (UDP-transferases) and retinal microvessel tissue subjected to complete or abbreviated protocols for BM purification. The results of these studies are expected to provide information on changes in RMV anionic sites in diabetes and insight into the etiologica link between enhanced platelet aggregation and the increased microvascular permeability of this disease, and to enhance our understanding of the defects in capillary filtration which contribute to diabetic retinopathy.